Compositional changes in subchondral bone and calcified cartilage in an equine cartilage defect model - a pilot study

Osteoarthritis (OA) is the most common degenerative joint disease [1] affecting the whole joint via cross-talk between the tissues [2]. It especially concerns articular cartilage (AC) and subchondral bone (SCB), the bone below AC, and calcified cartilage (CC), the mineralized tissue between AC and SCB. Diagnosis of the disease comes typically too late and is based on the degeneration of AC. In order to develop new strategies for treatments, early detection is essential. Therefore, pre- and early-OA compositional and structural markers of SCB and CC would be valuable. In this pilot study, we utilize Fourier transform infrared (FTIR) microspectroscopy to investigate the compositional alterations in CC and SCB using an equine cartilage defect model. Materials and methods: Samples: We induced unilateral cartilage defects in the radio- and middle carpal joints of adult Shetland ponies (N = 10) by scratching the AC with a sharpened arthroscopic probe (groove model). Three weeks post-surgery, ponies were subjected to an 8-week training protocol on a treadmill, followed by free exercise until the end of the study. At 39 weeks, ponies were euthanized and the joints were collected. FTIR: Maps of 3μm-thick sections of the samples including AC, CC, and SBC were imaged using 128x128 FPA detector with 5.5μm pixel size (Agilent Cary 670/620). Amide I (~1660cm-1, matrix), phosphate (~1030cm-1, mineral), and carbonate (~870cm-1) peak areas were determined. From these peak areas, mineral/matrix (M/M), carbonate/matrix (C/M), and carbonate/phosphate (C/P) ratios were calculated. Collagen maturity (XLR, 1660/1690cm-1), crystallinity (1030/1020cm-1), and acid phosphate substitution (APS, 1027/1096cm-1) were determined from intensity ratios. Statistics: Mann-Whitney U-test was used to test differences between control (N=5) and grooved (N=6) samples (torn sections and missing samples excluded). p<0.05 was considered significant. Results: Compositional (Fig 1) and mechanical parameters (data not shown) of AC were similar at the grooved and the control samples. However, in CC and SCB, the XLR, APS, and mineralization related parameters were significantly lower for the grooved samples (Fig 1). Discussion: Current results suggest that the observed decrease in the mineralization (as manifested in phosphate content, crystallinity, M/M) and collagen maturity (XLR) is evident already at an early degenerative state. Similarly, a decrease in APS, an indicator of new bone formation, suggests decreased new mineral deposition. The decrease in SCB mineralization has earlier been shown to correlate with SCB sclerosis in advanced OA [3]. To conclude, the results propose that the potentially early-OA related compositional changes in SBC and CC can be detected sensitively using microspectroscopic methods.